期刊
JOURNAL OF CLINICAL MICROBIOLOGY
卷 54, 期 10, 页码 2470-2484出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JCM.00330-16
关键词
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类别
资金
- Medical Research Council, United Kingdom [MR/K01532X/1]
- Medical Research Foundation [C0365]
- Wellcome Trust [090532/Z/09/Z, 091663MA]
- PHE
- National Institute for Health Research (NIHR) Centre for Health Protection Research
- FP7 PATHSEEK grant
- MRC
- Oxford NHIR BRC
- Oxford Martin School
- Oxford Martin School, NIHR Biomedical Research Centre, Oxford, United Kingdom
- NIH [U19AI082630]
- NIHR UCL/UCLH Biomedical research consortium
- Wellcome Trust Centre for Human Genetics
- BBSRC [BBS/E/D/20241864] Funding Source: UKRI
- MRC [MC_UU_12014/1, MR/K01532X/1, MC_EX_UU_G1000717, MR/K010239/1, G0801822, MC_UU_12014/12] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BBS/E/D/20241864] Funding Source: researchfish
- Medical Research Council [MC_EX_UU_G1000717, MR/K010239/1, MC_UU_12014/1, MC_UU_12014/12, G0801822, MR/K01532X/1] Funding Source: researchfish
- Medical Research Foundation [C0365] Funding Source: researchfish
- National Institute for Health Research [14/02/17, NF-SI-0515-10005] Funding Source: researchfish
- Wellcome Trust [102789/Z/13/Z, 109965/Z/15/Z] Funding Source: researchfish
Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub) types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.
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